Peroxynitrite induces covalent dimerization of epidermal growth factor receptors in A431 epidermoid carcinoma cells

Irreversible tyrosine modifications by inflammatory oxidants such as peroxynitrite (ONOO-) can affect signal transduction pathways involving tyrosine phosphorylation. The epidermal growth factor receptor (EGFR), a member of the c-ErbB receptor tyrosine kinase family, is involved in regulation of epithelial cell growth and differentiation, and possible modulation of EGFR-dependent signaling by ONOO- was studied. Exposure of epidermoid carcinoma A431 cells to 0.1-1.0 mM ONOO- resulted in tyrosine nitration on EGFR and other proteins but did not significantly affect EGFR tyrosine autophosphorylation. A high molecular mass tyrosine-phosphorylated protein (approximately 340 kDa) was detected in A431 cell lysates after exposure to ONOO-, most likely representing a covalently dimerized form of EGFR, based on immunoprecipitation and/or immunoblotting with alpha-EGFR antibodies and co-migration with ligand-induced EGFR dimers cross-linked with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. Covalent EGFR dimerization by ONOO- probably involved intermolecular dityrosine cross-linking and was enhanced after receptor activation with epidermal growth factor. Furthermore, irreversibly cross-linked EGFR was more extensively tyrosine-phosphorylated compared with the monomeric form, indicating that ONOO- preferentially cross-links activated EGFR. Exposure of A431 cells to ONOO- markedly reduced the kinetics of tyrosine phosphorylation of a downstream EGFR substrate, phospholipase C-gamma1, which may be related to covalent alterations in EGFR. Alteration of EGFR signaling by covalent EGFR dimerization by inflammatory oxidants such as ONOO- may affect conditions of increased EGFR activation such as epithelial repair or tumorigenesis.     

Measles virus infection induces terminal differentiation of human thymic epithelial cells

Measles virus infection induces a profound immunosuppression that may lead to serious secondary infections and mortality. In this report, we show that the human cortical thymic epithelial cell line is highly susceptible to measles virus infection in vitro, resulting in infectious viral particle production and syncytium formation. Measles virus inhibits thymic epithelial cell growth and induces an arrest in the G0/G1 phases of the cell cycle. Moreover, we show that measles virus induces a progressive thymic epithelial cell differentiation process: attached measles virus-infected epithelial cells correspond to an intermediate state of differentiation while floating cells, recovered from cell culture supernatants, are fully differentiated. Measles virus-induced thymic epithelial cell differentiation is characterized by morphological and phenotypic changes. Measles virus-infected attached cells present fusiform and stellate shapes followed by a loss of cell-cell contacts and a shift from low- to high-molecular-weight keratin expression. Measles virus infection induces thymic epithelial cell apoptosis in terminally differentiated cells, revealed by the condensation and degradation of DNA in measles virus-infected floating thymic epithelial cells. Because thymic epithelial cells are required for the generation of immunocompetent T lymphocytes, our results suggest that measles virus-induced terminal differentiation of thymic epithelial cells may contribute to immunosuppression, particularly in children, in whom the thymic microenvironment is of critical importance for the development and maturation of a functional immune system.     

Epidermal growth factor modulates fetal thymocyte growth and differentiation

In the present study, we used the fetal organ culture (FTOC) technique in order to study a putative effect of epidermal growth factor (EGF) on the thymus ontogeny. Functional EGF receptors and more recently the EGF molecule itself, respectively, on the membrane of epithelial components of thymic stroma and on a few thymocytes in adult thymus, had been reported in the literature. We could observe a dose-dependent decrease in cellularity and a progressive retention of thymocytes in the double-negative (CD4-/CD8-) stage of differentiation when exogenous EGF was added. Epidermal growth factor interfered with both fetal stroma growth and thymocyte development at a precise moment, that is, in the passage from double-negative to the double-positive (CD4+/CD8+) stage. After a 7-day FTOC in the presence of EGF, most cells recovered were Thy-1.2+, c-kit+, TSA1-/int, CD3-, and one of CD44high/CD25int, CD44-/CD25int, or CD44/CD25-. Some developed into gammadeltaTCR+ cells with a mature (CD3+) phenotype, but not into alphabetaTCR+ thymocytes. It seems that EGF addition makes the cultures "nonpermissible" for alphabetaTCR+ thymocyte generation. We report here the presence of a high Mr "EGF-like" molecule on the membrane of fetal thymocytes, which role in the observed effects is under investigation. Further biochemical characterization of this molecule is still required, because its presence was only evidenced on the basis of its antigenicity.     

EGF-receptor tyrosine kinase inhibition induces keratinocyte growth arrest and terminal differentiation

Epidermal keratinocyte growth and differentiation are regulated by specific families of growth factors and receptors. Peptide growth factors of the epidermal growth factor family stimulate proliferation of clonal density human keratinocytes and suppress markers of terminal differentiation in confluent cultures of human keratinocytes. We present evidence that selected inhibitors of activation of the type I human epidermal growth factor receptor (EGFR or HER-1), namely, neutralizing monoclonal antibody to HER-1/EGFR and the specific tyrosine kinase inhibitor PD 153035, potently inhibit proliferation of human keratinocytes in autonomously replicating subconfluent cultures. Coupled to growth arrest is the suppression of HER-1 tyrosine autophosphorylation in inhibitor-treated human keratinocytes. Proliferation and tyrosine autophosphorylation are initially reversible following removal of the inhibitor and restimulation of cells with epidermal growth factor. Sustained inactivation of HER-1 in autonomously replicating cultures of human keratinocytes induces expression of keratin 1 and keratin 10 genes, early markers of terminal differentiation. Reversal of growth inhibition by epidermal growth factor suppresses keratin 1 and keratin 10 expression. These results demonstrate that human keratinocyte terminal differentiation as well as proliferation are mediated by HER-1. Co-expression of autocrine epidermal growth factor-related ligands as well as HER-1 by human keratinocyte may function as part of the signal transduction network in epidermis to regulate cell number, replication rate, and terminal differentiation.     

Epidermal growth factor enhances transcription of human arachidonate 12-lipoxygenase in A431 cells

BACKGROUND/AIMS: Prognostic studies on cirrhosis are needed, since several attempts to obtain better survival predictors than the empirically derived Child-Pugh's score have failed. METHODS: Four hundred and ninety-four consecutive in-patients with cirrhosis at the first episode of decompensation (ascites, jaundice, encephalopathy) and/or of digestive haemorrhage from ruptured oesophageal varices were followed from admission (1983-1989) to 1993, studying the relationship between 26 prognostic variables and survival. Three prognostic models were constructed using Cox's regression model and the Receiver Operating Characteristic (ROC) analysis was used to compare their predictive ability. RESULTS: During follow-up 351 patients died (median cumulative survival 1.82 years). Child-Pugh's score (explicative variable of the first Cox's model), albumin and encephalopathy among the 5 Child-Pugh variables (second model), and oesophageal varices haemorrhage and 3 biochemical indexes among the 7 significant variables on univariate analysis (third model) correlated with survival. The area under the ROC curve of the first model did not significantly differ from that for the other 2 models. CONCLUSIONS: The Child-Pugh's score is still the best and simplest index for assessing the prognosis of liver cirrhosis.     

Epidermal growth factor receptors and their ligands in non-small-cell lung carcinoma

The study was carried out in 178 women without grave obstetrical or extragenital diseases. In group 1 labor pain was relieved by prolonged epidural anesthesia with 2% lidocaine solution (2-2.5 mg/kg), in group 2 prolonged epidural anesthesia with 1% lidocaine solution (1 mg/kg) and 0.01% clofelin (1 microgram/kg) was administered. Central hemodynamics, heart rhythm, external respiration function, uterine contractility, and fetal intrauterine status were assessed. The findings indicate that none of the methods had a negative impact on the vital parameters of women and newborns at any stage of anesthesia. However, a combination of epidural clofelin (1 microgram/kg) with lidocaine permits an appreciable decrease in the doses of both drugs without decreasing the efficacy of anesthesia. This method has a favorable effect on the course of labor: the mouth of the womb opens sooner at a lower uterine activity and there are no negative effects on the fetus and newborn.     

Insulin-like growth factor binding protein production in human follicular thyroid carcinoma cells

OBJECTIVE: The study investigated the value of using national or regional data bases to examine care in a specific hospital. DATA SOURCES: The following data sources were included: (1) the results of the 1992 HCFA analysis of the index hospital for patients hospitalized in fiscal year 1990; (2) the 1989 Medicare Provider Analysis and Review (MEDPAR) file; and (3) clinical information from bypass surgery patients in Wisconsin and from the index hospital. PRINCIPAL FINDINGS: The assessment of the mortality rates in the index hospital for all conditions combined and for CABG patients differed depending on what data base was used and how the data were analysed. The national data were most useful in establishing that the coding practices for all patients and the mortality rate for intra-aortic balloon patients differed between the index hospital and other hospitals. The regional clinical data base for bypass surgery patients was used to establish that the high mortality rates for intra-aortic balloon patients were due to patient selection. CONCLUSIONS: National claims data must be analysed carefully before applying results to an individual hospital. Even a careful analysis is more for raising questions about care at a specific hospital rather than for reaching definitive conclusions.     

EGFR blockade by tyrosine kinase inhibitor or monoclonal antibody inhibits growth, directs terminal differentiation and induces apoptosis in the human squamous cell carcinoma HN5

Xenotransplantation of human cells into immunodeficient mice has been used to develop models of human haemopoiesis and lymphoid cell function. However, the utility of existing mouse strains can be limited by shortened life-spans, spontaneous production of functional lymphocytes with ageing, and residual innate immunity leading to variable levels of engraftment. Mice with a deletion of the common cytokine receptor gamma chain (gamma c) gene have reduced numbers of peripheral T and B lymphocytes, and absent natural killer cell (NK) activity. A genetic cross with a recombinase activating gene 2 (RAG2)-deficient strain produced mice doubly homozygous for the gamma c and RAG2 null alleles (gamma c-/RAG2-). These mice have a stable phenotype characterized by the absence of all T lymphocyte. B lymphocyte and NK cell function. Injection of human B-lymphoblastoid cells resulted in earlier fatal metastatic lymphoproliferative disease than in NOD/LtSz-scid controls. This was particularly evident in animals injected intravenously, possibly because of residual NK activity in NOD/LtSz-scid mice. Levels of engraftment with peripheral-blood-derived human lymphocytes were also increased and associated with higher CD4/CD8 ratios. These findings demonstrate that this new strain of immunodeficient mice has significant advantages over existing strains for engraftment of human cells, and may be useful for study of adoptive immunotherapy and novel therapies for GvHD and HIV infection.     

Calpain-calpastatin interactions in epidermoid carcinoma KB cells

We examined the activation of mu-calpain in human epidermoid carcinoma KB cells following a rise in intracellular calcium concentration using antibodies specifically recognizing different activation states of mu-calpain. KB cells possess calpastatin activity in large excess of calpain activity as analyzed by ion exchange HPLC. Stimulation of the cells with a calcium ionophore, ionomycin, caused production of the autolytic intermediate form (M(r) = 78 k) of mu-calpain derived from the preautolysis form (80 k), while the fully autolyzed postautolysis form (76 k) remained below detectable levels at all times. The appearance of the autolytic intermediate paralleled limited proteolysis of the membrane-associated calpastatin fractions (110 k and 106 k); the resulting fragments (68 k and 45 k) were released into the cytosol. Both the production of the autolytic mu-calpain intermediate and the limited proteolysis of calpastatin in cell lysates in the presence of calcium were inhibited by a synthetic calpastatin peptide, indicating that proteolysis of calpastatin was indeed catalyzed by calpain and that the autolytic intermediate may have exerted the proteolytic activity. Furthermore, mu-calpain autolysis and calpastatin degradation, upon ionomycin treatment, were both augmented by epidermal growth factor (EGF). These results suggest that calpastatin serves not only as an inhibitor but also as a substrate for calpain at cell membranes and that intracellular conditions associated with the cell cycle may affect the activation of mu-calpain.     

Fibroblast growth factor receptor expression reflects cellular differentiation in human oral squamous carcinoma cell lines

This study examined the expression of fibroblast growth factor receptor 2 (FGFR 2) splice variants, IIIb and IIIc, in normal and malignant human oral keratinocytes and in normal oral fibroblasts by RT-PCR using both exon-specific primers and primers common to both FGFR 2 isoforms. Fibroblasts expressed exclusively FGFR 2/IIIc whilst the normal and malignant keratinocytes co-expressed FGFR 2/IIIb and FGFR 2/IIIc. Well-differentiated keratinocytes expressed proportionally more FGFR 2/IIIb than IIIc whereas the poorly-differentiated cells expressed more FGFR 2/IIIc than IIIb. The normal and malignant keratinocytes, but not fibroblasts, expressed an additional amplification product, which consisted of both IIIb and IIIc of FGFR 2 joined by an extra base pair and with the intronic sequence removed. The results indicate that the expression of FGFR 2 isoforms reflects the degree of cellular differentiation in normal and malignant human oral keratinocytes and that receptor complexes of FGFR 2/IIIb and IIIc may regulate ligand-receptor interactions.     

Association of vascular endothelial growth factor expression with intratumoral microvessel density and tumour cell proliferation in human epidermoid lung carcinoma

Vascular endothelial growth factor (VEGF) expression, vascularisation and tumour cell proliferation were analysed in 91 human epidermoid lung carcinomas using immunohistochemistry. A polyclonal anti-VEGF antibody was used for VEGF expression, a polyclonal antibody directed against human von Willebrand factor (factor VIII) to identify blood vessels and the proliferating cell nuclear antigen (PCNA) as a marker for proliferating cells. Positive staining for VEGF was obtained in 54 out of 91 cases (59%), the number of blood vessels varied from zero to 64 counts (mean 9.4) and the proportion of PCNA-positive cells varied from 1.3% to 72.1% (mean 25.2%). The mean PCNA labelling index and mean microvessel count in VEGF-positive tumours were significantly higher than those in VEGF-negative tumours (Wilcoxon rank sum test, P<0.0001; p<0.05). In addition, PCNA labelling index significantly increased with increasing VEGF expression (Jonckheere test, P<0.0001). In contrast, no association was found between PCNA labelling index and tumour vascularity (r=0.07, P=0.48). The close correlation of VEGF expression with tumour cell proliferation and microvessel density suggests that VEGF acts both as an autocrine growth factor and as stimulator for angiogenesis. However, tumour cell proliferation and microvessel growth and/or density may be regulated by separate mechanisms.     

Enhanced growth inhibition and differentiation of fluorodeoxyuridine-treated human colon carcinoma cells by phenylbutyrate

The effect of phenylbutyrate (PB), a nontoxic differentiation inducer, in human colon carcinoma cell lines treated with 5-fluorodeoxyuridine (FUdR) was evaluated. Two HT-29 human colon carcinoma subclones (U4 well differentiated and U9 poorly differentiated) were equally growth inhibited by 16 h of FUdR (0.2 microM) treatment but recovered cell growth in 3-6 days after the removal of FUdR. PB as a single agent had minimal effect on cell growth, but after FUdR treatment, PB inhibited cell growth for 12 days. The inhibition of cell growth in FUdR-treated cells by PB was more sustained in U4 than U9 cells and was associated with an increased and sustained expression of p21waf1 protein, secretion of transforming growth factor beta1, mediators of p53-dependent or -independent G1 cell cycle arrest, and an increase in the alkaline phosphatase activity as well, considered a marker of differentiation in colon carcinoma cells. These effects of PB were seen only in FUdR-pretreated cells because PB alone had minimal effect on the expression of these genes. The sequential use of FUdR followed by PB in patients with colon carcinoma should be explored because two subclones of HT29, irrespective of their state of differentiation, respond to this clinically achievable regimen.     

Inhibition of epidermal growth factor receptor kinase induces protease-dependent apoptosis in human colon cancer cells

BACKGROUND & AIMS: The epidermal growth factor receptor (EGFR) is under investigation as a therapeutic target for cancers. Colon cancer cell lines are variably dependent on autocrine stimulation of EGFR. We therefore examined the effects of a selective EGFR tyrosine kinase inhibitor, PD 153035, on proliferation and survival of five colon cancer cell lines whose autonomous proliferation is either EGFR ligand dependent or EGFR ligand independent. METHODS: Effects of inhibitors were screened by MTS growth assays, [3H]thymidine incorporation, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling assay, fluorescence microscopy, immunoblotting, and in vitro protease assays. RESULTS: PD 153035 caused dose-dependent cytostasis (200 nmol/L to 1 micromol/L) and apoptosis (>10 micromol/L) in ligand-dependent cell lines and caused variable apoptosis (>10 micromol/L) but no cytostasis in ligand-independent cell lines. Apoptosis induced by 10 micromol/L PD 153035 was not associated with induction of p53 protein expression but was accompanied by activation of caspases that cleave poly(ADP-ribose) polymerase, lamin B1, and Bcl-2. Inhibition of caspase 3-like protease activity by DEVD-fluoromethylketone significantly delayed the onset of PD 153035-induced apoptosis. CONCLUSIONS: The EGFR tyrosine kinase inhibitor PD 153035 induces cytostasis and caspase-dependent apoptosis in EGFR ligand-dependent colon cancer cell lines. These observations encourage further investigation of EGFR tyrosine kinase inhibitors for treatment of colorectal neoplasms.     

Epidermal growth factor receptor-mediated motility in fibroblasts

Cell motility is induced by many growth factors acting through cognate receptors with intrinsic tyrosine kinase activity (RPTK). However, most of the links between receptor activation and the biophysical processes of cell motility remain undeciphered. We have focused on the mechanisms by which the EGF receptor (EGFR) actuates fibroblast cell motility in an attempt to define this integrated process in one system. Our working model is that divergent, but interconnected pathways lead to the biophysical processes necessary for cell motility: cytoskeleton reorganization, membrane extension, formation of new adhesions to substratum, cell contraction, and release of adhesions at the rear. We postulate that for any given growth factor some of the pathways/processes will be actively signaled and rate-limiting, while others will be permissive due to background low-level activation. Certain couplings have been defined, such as PLCgamma and actin modifying proteins being involved in cytoskeletal reorganization and lamellipod extension and MEK being implicated in detachment from substratum. Others are suggested by complementary investigations in integrin-mediated motility, including rac in membrane protrusion, rho in new adhesions, myosin II motors in contraction, and calpain in detachment, but have yet to be placed in growth factor-induced motility. Our model postulates that many biochemical pathways will be shared between chemokinetic and haptokinetic motility but that select pathways will be activated only during RPTK-enhanced motility.     

Epidermal growth factor receptor ectodomain in the urine of patients with squamous cell carcinoma

Female Sprague-Dawley rats were ovariectomized (OVX) or sham-operated at 3 months of age and maintained untreated for 1 year after surgery. Baseline control and OVX rats were killed at the beginning of treatment when the rats were 15 months of age and 1 year postovariectomy. The remaining rats were treated with hPTH 1-34 (80 micrograms/kg BW, 5 days/week) or vehicle for 10 weeks. Quantitative bone histomorphometry was performed on undecalcified longitudinal sections of the proximal femur from each rat. Baseline OVX rats exhibited cancellous and cortical osteopenia at the femoral neck as their mean cancellous bone volume and cortical width were significantly decreased compared to the means for baseline control rats. In addition, baseline OVX rats had increased osteoblast and osteoclast surfaces and a greater cancellous bone formation rate than baseline control rats. OVX rats remained osteopenic with no further bone loss from the femoral neck after 10 weeks of vehicle treatment. In contrast, cancellous bone volume and cortical width in OVX rats treated with PTH were increased to the level of vehicle-treated control rats. The hormone restored lost bone in the femoral neck of OVX rats by markedly stimulating both cancellous and cortical bone formation. These histomorphometric findings in concert with recent biomechanical studies of bone strength indicate that the femoral neck of aged OVX rats is a promising sample site for studies of the prevention and treatment of bone loss induced by estrogen depletion.     

Epidermal growth factor family and renal cell carcinoma: expression and prognostic impact

OBJECTIVE: Expression and prognostic impact of some exponents of the epidermal growth factor (EGF) family in renal cell carcinoma (RCC) were examined. MATERIALS AND METHODS: EGF, transforming growth factor-alpha (TGF-alpha), EGF receptor (EGF-R), and c-erb B-2 were determined immunohistochemically in formalin-fixed paraffin-embedded tumor samples of 30 patients with locally confined RCCs. The prognostic significance of these growth factors and their receptors as well as of tumor stage and malignancy grade was examined with respect to survival and tumor recurrence by following up the fate of the patients after nephrectomy (mean follow-up time 5.2 years). RESULTS: The members of the EGF family and their receptors studied were expressed to a variable degree in all RCCs investigated. However, using log-rank tests in Kaplan-Meier plots only tumor stage (p < 0.0007) and malignancy grade (p < 0.007) but none of the growth factors or receptors studied (p > 0.05, respectively) exhibited prognostic significance with respect to both survival and disease-free period. On the contrary, there was a significant correlation between EGF and TGF-alpha (p < 0.001), EGF and EGF-R (p = 0.028), EGF-R and c-erb B-2 (p = 0.0009), and-inversely related-between TGF-alpha and tumor stage (p = 0.047) and between EGF-R and malignancy grade (p = 0.03). The coexpression of the factors studied also showed no prognostic relevance. CONCLUSION: The expression of these members of the EGF family seems not to bear evaluable prognostic information for clinical use in the case of RCC.     

Leukemia inhibitory factor induces apoptosis and proliferation of human carcinoma cells through different oncogene pathways

The aim of this study was to investigate the effects of a high ambient pressure of He on vascular contraction induced by noradrenaline and to distinguish the effects of ambient pressure per se from those of increased pressure of inert gas. Rings of thoracic aorta were isolated from male Wistar rats. Isometric tension was measured in preparations exposed to 7.1 MPa (absolute pressure) of He. Dose-response curves for noradrenaline and contractions elicited by 120 mM KCl were compared with time-matched experiments performed at atmospheric pressure. The same protocol was also carried out under 7.1 MPa of N2. At the high pressure of He, the contraction elicited by noradrenaline was increased with no change in the response to K(+)-evoked depolarization. The tension developed in response to noradrenaline also increased under 7.1 MPa of N2 but the effects were less marked than during the He experiments. Moreover, the response to KCl was reduced in this circumstance. Hyperbaric conditions enhance the noradrenaline-induced contraction of rat aorta in vitro. This effect probably results from an action of pressure per se on activation of adrenoceptors. However, the hyperbaric-induced increase in vascular smooth muscle contraction is partially counteracted by high pressures of inert gases (N2, but also probably He), which impair the efficiency of the contractile machinery.     

Autocrine actions of endothelin-1 as a growth factor in human ovarian carcinoma cells

The production of endothelin 1 (ET-1) and its receptor-mediated actions on calcium signaling and growth responses were analyzed in human ovarian carcinoma cells. Immuno-reactive ET-1 was released from three of four ovarian tumor cell lines as a function of time in amounts ranging from 56 to 74 fmol/10(6) cells. Reverse-phase HPLC and radioimmuno-assay of conditioned media from tumor cells revealed a single peak coeluting with authentic ET-1. Radioligand binding studies showed that the ET-1-producing cell lines also expressed high-affinity ETA receptors (Kd < 0.1 nM) that ranged in abundance from 2,600 to 43,600 sites/cell. In fura-2-loaded ovarian carcinoma cells, ET-1 induced dose-dependent increases in cytoplasmic Ca2+ concentration. ET-1 also stimulated thymidine incorporation in the three cell lines that expressed ET receptors. In OVCA 433 cells, BQ 123 inhibited the stimulatory actions of ET-1 on thymidine incorporation and cell proliferation, and substantially reduced the basal growth rate of unstimulated ovarian tumor cells. These results demonstrate that ET-1 is produced in ovarian cancer cells and acts as an autocrine growth factor on ETA receptors to stimulate calcium signaling and proliferative responses. Such findings suggest that ET-1 participates in the progression of neoplastic growth in certain ovarian tumors.